Gas chromatographymass spectrometry (GCMS) has been used for urinary steroid analysis for over 50 years. The process of hydrolysis to release steroids from their conjugates is routinely performed using commercially available snail Helix-Pomatia sulfatase (HP). However, as this is a naturally occurring enzyme its production process results in batch variation and so each batch requires experimental validation to adjust for the difference in efficiency of the enzymatic hydrolysis. Thus, a more efficient and consistent solution would be desirable. Steroid conjugates were hydrolysed firstly in 1 ml of pooled healthy male urine. The urine underwent C18 solid-phase-extraction (SPE), followed by enzymatic hydrolysis using either an optimised protocol for HP deconjugation (Sigma-Aldrich, UK) or a synthetic β-Glucuronidase/Sulfatase mix (BGS) enzyme (Kura Biotec, Puerto Varas, Chile). Then a second SPE followed by a two-step methyloxime-trimethylsilyl derivatization (MO-TMS) process. Samples were then analysed by GCMS using an Agilent 5975 instrument (SIM mode) for steroid identification and quantification, followed by comparison of urinary steroid outputs from the two different enzyme hydrolysis methods. The BGS hydrolysis method involved a shorter incubation (30-min) than HP (3-h). HP and BGS performed equally for all examined glucuronides. For monosulfates, BGS showed a lower deconjugation ability ranging from 35 to 60% compared to the HP. Unlike HP, BGS did not efficiently hydrolyse 21-sulphated hydroxyls or 16β- and-17β sulphates. Under recommended hydrolysis conditions BGS had reduced ability to deconjugate delta-5-sulphated steroids; approximately a third of that observed with HP. The new synthetic-combined BGS performed well for glucuronides but further optimisation of enzymatic hydrolysis conditions is required to maximise delta-5 steroid recovery.